Bottom Line:
We previously reported that interleukin-1ß (IL-1ß) can downregulate MITF-M levels.Notably, we also observed a strong negative correlation between MITF-M and miR-155 levels in a mouse model of melanoma.Taken together, our results indicate that MITF-M downregulation by inflammatory stimuli might be partly due to miR-155 upregulation.

ABSTRACTLoss of expression of surface antigens represents a significant problem for cancer immunotherapy. Microphthalmia-associated transcription factor (MITF-M) regulates melanocyte fate by driving expression of many differentiation genes, whose protein products can be recognized by cytolytic T lymphocytes. We previously reported that interleukin-1ß (IL-1ß) can downregulate MITF-M levels. Here we show that downregulation of MITF-M expression by IL-1ß was paralleled by an upregulation of miR-155 expression in four melanoma lines. We confirmed that miR-155 was able to target endogenous MITF-M in melanoma cells and demonstrated a role for miR-155 in the IL-1ß-induced repression of MITF-M by using an antagomiR. Notably, we also observed a strong negative correlation between MITF-M and miR-155 levels in a mouse model of melanoma. Taken together, our results indicate that MITF-M downregulation by inflammatory stimuli might be partly due to miR-155 upregulation. This could represent a novel mechanism of melanoma immune escape in an inflammatory microenvironment.

pone.0122517.g004: The repression of MITF-M by IL-1ß can be prevented at an early time point by the transfection of an AntagomiR specific to miR-155.LB2201-MEL cells were transfected and incubated for 24h with an AntagomiR specific for miR-155 or a control AntagomiR (150 nM) and then treated with IL-1ß (10 ng/ml) for 2h, 3h, 4h or 5h. This experiment is representative of 3 independent experiments.

Mentions:
To confirm that endogenous miR-155 contributes to the repression of MITF-M expression by IL-1ß, we used antisense inhibitors specific for miR-155. First, we transfected LB2201-MEL cells with either an inhibitor of miR-155 (AntagomiR anti-miR-155) or a control inhibitor. After 24h, we treated the cells with IL-1ß and analyzed the abundance of MITF-M protein 2h, 3h, 4h or 5h later by Western Blot. The repression of MITF-M expression induced by IL-1ß appeared to be delayed in the presence of the anti-miR-155, with the strongest effect after 2h of treatment with IL-1ß (Fig 4). This indicates that endogenous miR-155 contributes to the IL-1ß-induced downregulation of MITF-M. However, the lack of effect of anti-miR-155 at later time points suggests that the inhibitor might not be sufficiently effective at high miR-155 levels observed at later time points. Alternatively, there might be other mechanisms that are more important for IL-1ß-induced downregulation of MITF-M at later time points.

pone.0122517.g004: The repression of MITF-M by IL-1ß can be prevented at an early time point by the transfection of an AntagomiR specific to miR-155.LB2201-MEL cells were transfected and incubated for 24h with an AntagomiR specific for miR-155 or a control AntagomiR (150 nM) and then treated with IL-1ß (10 ng/ml) for 2h, 3h, 4h or 5h. This experiment is representative of 3 independent experiments.

Mentions:
To confirm that endogenous miR-155 contributes to the repression of MITF-M expression by IL-1ß, we used antisense inhibitors specific for miR-155. First, we transfected LB2201-MEL cells with either an inhibitor of miR-155 (AntagomiR anti-miR-155) or a control inhibitor. After 24h, we treated the cells with IL-1ß and analyzed the abundance of MITF-M protein 2h, 3h, 4h or 5h later by Western Blot. The repression of MITF-M expression induced by IL-1ß appeared to be delayed in the presence of the anti-miR-155, with the strongest effect after 2h of treatment with IL-1ß (Fig 4). This indicates that endogenous miR-155 contributes to the IL-1ß-induced downregulation of MITF-M. However, the lack of effect of anti-miR-155 at later time points suggests that the inhibitor might not be sufficiently effective at high miR-155 levels observed at later time points. Alternatively, there might be other mechanisms that are more important for IL-1ß-induced downregulation of MITF-M at later time points.

Bottom Line:
We previously reported that interleukin-1ß (IL-1ß) can downregulate MITF-M levels.Notably, we also observed a strong negative correlation between MITF-M and miR-155 levels in a mouse model of melanoma.Taken together, our results indicate that MITF-M downregulation by inflammatory stimuli might be partly due to miR-155 upregulation.

ABSTRACTLoss of expression of surface antigens represents a significant problem for cancer immunotherapy. Microphthalmia-associated transcription factor (MITF-M) regulates melanocyte fate by driving expression of many differentiation genes, whose protein products can be recognized by cytolytic T lymphocytes. We previously reported that interleukin-1ß (IL-1ß) can downregulate MITF-M levels. Here we show that downregulation of MITF-M expression by IL-1ß was paralleled by an upregulation of miR-155 expression in four melanoma lines. We confirmed that miR-155 was able to target endogenous MITF-M in melanoma cells and demonstrated a role for miR-155 in the IL-1ß-induced repression of MITF-M by using an antagomiR. Notably, we also observed a strong negative correlation between MITF-M and miR-155 levels in a mouse model of melanoma. Taken together, our results indicate that MITF-M downregulation by inflammatory stimuli might be partly due to miR-155 upregulation. This could represent a novel mechanism of melanoma immune escape in an inflammatory microenvironment.